Multi-locus sequence analysis unveils a novel genus of filarial nematodes associated with ticks in French Guiana

Filarial nematodes of the Dipetalonema lineage include tick-borne filarioids that infect both domestic and wild vertebrate hosts, but they remain understudied in many cases. In this study, we conducted a molecular characterization of a Dipetalonema-like filarioid (DLF) recently identified in two tick species in French Guiana, South America. While the cox1 mitochondrial gene was the sole marker initially sequenced for describing DLF, its classification and phylogenetic relationship with other members of the Dipetalonema lineage were unclear. Therefore, we better characterized DLF through the sequencing of six additional gene markers and conducted phylogenetic analyses. Based on this multi-locus typing scheme, DLF exhibited significant divergence from known genera and species of filarioids, or other sequences available in public databases, suggesting its potential classification as a novel genus within the Dipetalonema lineage. Phylogenetic analyses further unveiled a close evolutionary relationship between DLF and all other filarioids associated with Acari (ticks and mites) within a robust monophyletic subclade in the Dipetalonema lineage. Overall, these findings confirm the existence of a specialized, Acari-borne group of filarioids and underscore the need for comprehensive investigations into their epidemiology and potential impact on animal health.

In a recent survey of ticks in French Guiana, South America, molecular analysis and phylogenetic studies revealed the presence of novel filarioids belonging to the Dipetalonema lineage in several tick species [17].Based on cytochrome c oxidase subunit I (cox1) mitochondrial gene sequences, all but one of these filarioids are distinct to already known species of Dipetalonema lineage.Indeed, in the Cayenne tick Amblyomma cajennense (Fabricius, 1787) and in the opossum tick Ixodes luciae Sénevet, 1940, one filarioid, provisionally named Dipetalonema-like (DLF hereafter), showed a cox1 gene sequence substantially divergent from other species and genera of the Dipetalonema lineage [17].DLF could be of health concern since it was detected in A. cajennense [17], the predominant tick species biting humans in South America [16].This feature may not apply to I. luciae, as it is a specialized tick species with a primary feeding preference for opossums [16].However, while DLF has been detected in 6% of field specimens of A. cajennense [17], no further data are currently available on this filarioid.
In this study, we conducted an extended molecular characterization of DLF previously detected in A. cajennense and I. luciae in French Guiana.The cox1 gene sequence was the only genetic marker used for its description [17], but this marker exhibits limited resolution for inferring the evolutionary history in the family Onchocercidae [36].Using infected field specimens of A. cajennense and I. luciae, we thus characterized DLF through the sequencing of six additional genes (MyoHC, hsp70, rbp1, 12S rRNA, 28S rRNA, and 18S rRNA) previously used for inferring the Onchocercidae phylogeny [36].We further examined their genetic proximity with other filarioid species, including all known members of the Dipetalonema lineage, under a phylogenetic framework.

Tick collection
A collection of 10 DNA templates from A. cajennense (n = 8) and I. luciae (n = 2) infected by DLF was used for the present analysis.All templates were obtained from field specimens collected on vegetation through flagging (questing ticks) or on opossums (engorged ticks) in French Guiana in 2016 and 2017 (Table 1).Each DNA template was obtained from individual extraction of tick whole body using a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany), following manufacturer instructions.For each DNA template, infection by DLF had previously been confirmed through cox1 gene sequencing [17].Use of the genetic resources was approved by the French Ministry of the Environment under reference #TREL19028117S/156, in compliance with the Access and Benefit Sharing procedure implemented by the Loi pour la Reconquête de la Biodiversité.

Multi-locus typing of the Dipetalonema-like filarioid
Fragments of six genes (MyoHC, hsp70, rbp1, 12S rRNA, 28S rRNA, and 18S rRNA) were amplified using simple, seminested or nested PCR assays adapted from Lefoulon et al. [36].Gene features, primers and PCR conditions are detailed in Table S1.Simple PCR amplifications were performed in a total volume of 25 lL containing ca. 20 ng of genomic DNA, 8 mM of each dNTP (Thermo Scientific, Waltham, MA, USA), 10 mM of MgCl 2 (Thermo Scientific), 7.5 lM of each of the internal primers, 2.5 lL of 10ÂPCR buffer (Thermo Scientific), and 1.25 U of Taq DNA polymerase (Thermo Scientific).Nested and semi-nested PCR amplifications were performed as follows: the first PCR run with the external primers was performed in a 10 lL volume containing ca. 20 ng of genomic DNA, 3 mM of each dNTP (Thermo Scientific), 8 mM of MgCl 2 (Roche Diagnostics), 3 lM of each primer, 1 lL of 10 Â PCR buffer (Roche Diagnostics), and 0.5 U of Taq DNA polymerase (Roche Diagnostics).A 1 lL aliquot of the PCR product from the first reaction was then used as a template for the second round of amplification.The second PCR was performed in a total volume of 25 lL and contained 8 mM of each dNTP (Thermo Scientific), 10 mM of MgCl 2 (ThermoScientific), 7.5 lM of each of the internal primers, 2.5 lL of 10 Â PCR buffer (Thermo Scientific), and 1.25 U of Taq DNA polymerase (Thermo Scientific).S1).To prevent possible contamination, first and second PCR runs were physically separated from one another, in entirely separate rooms.Negative (water) controls were included in each PCR assay.All PCR products were visualized through electrophoresis in a 1.5% agarose gel.All amplicons were purified and sequenced in both directions (EUROFINS, Luxembourg).Sequence chromatograms were cleaned with Chromas Lite (http://www.technelysium.com.au/chromas_lite.html), and alignments were performed using ClustalW, implemented in the MEGA software package (https://www.megasoftware.net/).New sequences obtained in this study were deposited in GenBank under accession numbers PP182382-PP182391 (MyoHC), PP182371-PP182380 (hsp70), PP182391-PP182401 (rbp1), PP196371-PP196380 (12S rRNA), PP196417-PP196426 (28S rRNA), and PP196384-PP196393 (18s rRNA).

Molecular phylogenetic analyses
Phylogenetic analyses were based on sequence alignments of the filarioid MyoHC, hsp70, rbp1, 12S rRNA, 28S rRNA, and 18S rRNA gene sequences obtained in this study.Analyses also included the filarioid cox1 gene sequences (GenBank accession numbers OR030080-OR030087, OR030094, and OR030095) previously obtained from the same A. cajennense and I. luciae specimens by Binetruy and Duron [17].Sequences of other filarioids obtained from GenBank, including representative members of the Dipetalonema lineage (Acanthocheilonema, Yatesia, Cercopithifilaria, Cruorifilaria, Litomosoides, and Dipetalonema) and of other filarial nematodes were also included in the phylogenetic analyses (Table S2).The Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/blast/Blast.cgi)was used to find additional sequences available on GenBank.The Gblocks program with default parameters was used to obtain non-ambiguous sequence alignments [22].Tree-based phylogenetic analyses were performed using maximum-likelihood (ML) analyses using the MEGA software package (https://www.megasoftware.net/).The evolutionary models that best fit the sequence data were determined using the Akaike information criterion.Clade robustness was assessed by bootstrap analysis using 1,000 replicates.We further conducted a phylogenetic network analysis based on uncorrected P distances using the Neighbor-net algorithm [21] implemented in SPLITSTREE [30].The resulting phylogenetic networks generalize the trees by allowing cross-connections between branches, which might display conflicting signals in the phylogenetic data set [21].

Multi-locus typing of the Dipetalonema-like filarioid
The DLF MyoHC, hsp70, rbp1, 12S rRNA, 28S rRNA, and 18S rRNA gene sequences were amplified from the 10 DNA templates (A.cajennense, n = 8; I. luciae, n = 2).All sequences were easily readable without double peaks, indicating a confident degree of primer specificity for filarioid PCR amplifications.On the basis of DNA sequencing, we characterized only one allele for each of the six genes.The DLF MyoHC, hsp70, rbp1, 28S rRNA, and 18S rRNA gene sequences were distinct from sequences available in public databases, and showed 83.2-98.9%pairwise nucleotide identities (depending on gene sequence) with other members of the Dipetalonema lineage, including Acanthocheilonema, Monanema, Yatesia, and Cercopithifilaria spp.(Table 2).Comparisons with filarioids other than members of the Dipetalonema lineage showed lower pairwise nucleotide identities for these gene sequences although the 12S rRNA sequences exhibited the highest pairwise nucleotide identities with filarioids of uncertain phylogenomic position (Table 2).
The ML and network analyses based on the concatenated dataset produced congruent phylogenetic trees with no major differences (Figs. 1 and 2).All phylogenetic reconstructions revealed a clustering of DLF with the genera Cercopithifilaria, Cruorifilaria, Litomosoides, Yatesia, Acanthocheilonema, Monanema, and Dipetalonema in a single monophyletic clade, the Dipetalonema lineage, distinct from other members of the family Onchocercidae.Phylogenetic reconstructions further revealed the division of the Dipetalonema lineage into two monophyletic subclades supported by high bootstrap values (Figs. 1 and 2): 1.The first subclade comprised DLF and members of the genera Acanthocheilonema, Monanema, Cercopithifilaria, Yatesia, Cruorifilaria, and Litomosoides.Within this subclade, DLF formed a branch substantially divergent from all other genera, but was more related to Acanthocheilonema species.Remarkably, all these filarioids are naturally associated with Acari, either with ticks (for DLF, Acanthocheilonema, Monanema, Cercopithifilaria, Yatesia, and Cruorifilaria), or with parasitic mites (for Litomosoides).
2. The second subclade comprised only Dipetalonema spp., which are filarioids specifically associated with biting midges.
As a result, the phylogenetic partitioning of the Dipetalonema lineage into two monophyletic subclades correlates with specialization for distinct types of arthropod vectors, Acari vs. dipterans.

Discussion
In this study, we show that DLF displays substantial differences in its gene sequences compared to known genera and species within the family Onchocercidae, as well as any other sequences available in public databases.This observation lends support to the hypothesis that it could be a novel genus of filarioid with the Dipetalonema lineage.Furthermore, phylogenetic analyses unveil a close evolutionary relationship between DLF and all filarioids associated with Acari (ticks and mites): these filarioids cluster together in a robust monophyletic subclade within the Dipetalonema lineage.These findings, consistent with earlier observations by Lefoulon et al. [36], suggest the presence of a monophyletic group of filarioids that has evolved a specialization for Acari as specific vectors.
Analyses of DNA gene sequence similarities and phylogenetics both confirm that DLF is divergent from other members of the Dipetalonema lineage.No morphological data are currently available for DLF but it may share morphological similarities with other members of the Dipetalonema lineage.Adults of these species have a long tail, a buccal capsule divided into two (or three) segments, more or less atrophied for specialized species, and a caudal extremity with two subterminal lappets [7,23,32].Interestingly, opossums could be vertebrate hosts of DLF since I. luciae is a specialized tick species that feeds primarily on opossums [16].Under this assumption, DLF may have previously been observed in opossums: previous studies have identified four filarioid species, all showing typical morphological features of the Dipetalonema lineage, i.e., Acanthocheilonema pricei, Cercopithifilaria didelphis, Skrjabinofilaria skrjabini, and Cherylia guyanensis, in South American opossums [8,27].However, only morphological data, and no molecular data, are currently available for these four filarioid species, which prevents us from concluding whether one of these already described species is a DLF.
The clustering of all Acari-associated filarioids in a monophyletic subclade, separate from those transmitted by blood-feeding dipterans, strengthens the conclusion that ticks serve as specific vectors for certain filarioids.It also implies that these filarioids are well adapted to tick physiology, life-cycle and behavior.Earlier experimental assays have confirmed that ticks are competent vectors of filarioids of the Dipetalonema lineage [3,5,20,33,39,40,43,48,51,52].These observations include the filarioid Cherylia guianensis, primarily isolated from a gray and black four-eyed opossum, which can normally develop in Ixodes ticks [8].Furthermore, the detection of DLF from questing (unfed) A. cajennense ticks that have already digested their previous blood meals, have further moulted, and are seeking vertebrates for their next blood meal suggests that A. cajennense can acquire and stably maintain infection through transstadial transmission [17], as also observed for other members of the Dipetalonema lineage [40,48].For animals, the risk of acquiring a DLF infection is currently unknown, but surveys of dogs and capybaras infected by other filarioids of the Dipetalonema lineage revealed skin issues, chronic polyarthritis, anemia, and kidney and pulmonary damage [18,19,25,26,28,41]. conclusion, ticks transmit a broader range of infectious agents than any other arthropod vector, but their role as vectors of filarioids is less well-documented.The recurring identification of the Dipetalonema lineage species in major tick genera on most continents [2,12,17,19,31,35,37,42,46,47] confirms that these are widespread but overlooked tick-borne parasites.Further research is needed to understand their pathogenicity, epidemiology, developmental cycles, and transmission mechanisms by ticks, including DLF in A. cajennense and I. luciae.

Table 2 .
Best nucleotide identities of DLF MyoHC, hsp70, rbp, 12S rRNA, 28S rRNA, and 18S rRNA gene sequences obtained in this study with sequences available in GenBank.